mesenchymal stem/ stromal cells metabolomic and bioactive … · 44 stromal cells secretion, while...

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1 (Title) Mesenchymal Stem/ Stromal Cells metabolomic and bioactive factors

2 profiles: a comparative analysis on the Umbilical Cord and Dental Pulp derived

3 Stem/ Stromal Cells secretome

4 (Running title) A comparative analysis on secretome profile of MSCs

5

6 Authors:

7 AR Caseiro 1,2,3,#, SS Pedrosa 1,2,#, G Ivanova 4, MV Branquinho 1,2, A Almeida 2,5, F Faria 6, I

8 Amorim 6,7, T Pereira 1,2, AC Maurício 1,2,*

9 # AR Caseiro e SS Pedrosa contributed equally and should both be considered as first authors

10

11 Affiliations:

12 1 Departamento de Clínicas Veterinárias, Instituto de Ciências Biomédicas de Abel Salazar (ICBAS),

13 Universidade do Porto (UP), Rua de Jorge Viterbo Ferreira, nº 228, 4050-313 Porto, Portugal.

14 2 Centro de Estudos de Ciência Animal (CECA), Instituto de Ciências, Tecnologias e Agroambiente da

15 Universidade do Porto (ICETA), Rua D. Manuel II, Apartado 55142, 4051-401, Porto, Portugal.

16 3 Escola Universitária Vasco da Gama (EUVG), Av. José R. Sousa Fernandes 197, Campus Universitário –

17 Bloco B, Lordemão, 3020-210 Coimbra, Portugal

18 4 REQUIMTE- LAQV, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do

19 Porto, Rua do Campo Alegre, 4169-007 Porto, Portugal.

20 5 Indústria Transformadora de Subprodutos - I.T.S, SA, Grupo ETSA, Rua Padre Adriano, n.º 61, Olivais do

21 Machio, 2660-119 Santo Antão do Tojal, Loures, Portugal

22 6 Departamento de Patologia e Imunologia Molecular, Instituto de Ciências Biomédicas de Abel Salazar

23 (ICBAS), Universidade do Porto (UP), Rua de Jorge Viterbo Ferreira, nº 228, 4050-313 Porto, Portugal.

24 7 i3S - Instituto de Investigação e Inovação da Universidade do Porto, Rua Alfredo Allen, 4200-135 Porto,

25 Portugal

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31

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32 Abstract

33 Mesenchymal Stem/ Stromal Cells assume a supporting role to the intrinsic mechanisms

34 of tissue regeneration, a feature mostly assigned to the contents of their secretome. A

35 comparative study on the metabolomic and bioactive molecules/factors content of the

36 secretome of Mesenchymal Stem/ Stromal Cells derived from two expanding sources:

37 the umbilical cord stroma and the dental pulp is presented and discussed. The metabolic

38 profile (Nuclear Magnetic Resonance Spectroscopy) evidenced some differences in the

39 metabolite dynamics through the conditioning period, particularly on the glucose

40 metabolism. Despite, overall similar profiles are suggested. More prominent differences

41 are highlighted for the bioactive factors (Multiplexing Laser Bear Analysis), in which

42 Follistatin, Growth Regulates Protein, Hepatocyte Growth Factor, Interleukin-8 and

43 Monocyte Chemotactic Protein-1 dominate in Umbilical Cord Mesenchymal Stem/

44 Stromal Cells secretion, while in Dental Pulp Stem/ Stromal Cells the Vascular

45 Endothelial Growth Factor-A and Follistatin are more evident. The distinct secretory

46 cocktail did not result in significantly different effects on endothelial cell populations

47 dynamics including proliferation, migration, tube formation capacity and in vivo

48 angiogenesis, or in chemotaxis for both Mesenchymal Stem/ Stromal Cells populations.

49

50 1. Introduction

51 Mesenchymal Stem/ Stromal Cells (MSCs) are at the forefront of research for the

52 development of cell-based therapies, due to their capacity to self-renew and differentiate

53 into several cell types, to secrete soluble factors with paracrine actions, as well as due

54 to their immunosuppressive and immunomodulatory properties [1-5].

55 MSCs have been described to reside on nearly every body tissue [6-8] since Friedenstein

56 and colleagues firstly described the bone marrow derived population [9, 10].



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57 Currently, the umbilical cord stroma (Whärton jelly) and the dental pulp may come to

58 gain ground as sources for MSCs-based therapies, due to the non/ minimally invasive

59 and ethically accepted collection procedures (umbilical cords and extracted healthy teeth

60 were previously considered medical waste), as well as for the increasingly available

61 private and public banking options worldwide. Human MSCs characteristics have been

62 defined by the Mesenchymal and Tissue Stem Cell Committee of the International

63 Society for Cellular Therapy [2], being plastic-adherent when maintained in standard

64 culture conditions, expressing cluster of differentiation (CD) 105, CD73 and CD90, and

65 lacking expression of CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR

66 surface molecules. Finally, the MSCs must be able to differentiate in vitro into

67 osteoblasts, adipocytes and chondroblasts, in the presence of adequate differentiation

68 media [11].

69 The first evidence of the MSCs contribution to the healing processes was assigned to

70 their specific differentiation skills, replacing the damaged native cells in their functions

71 [12, 13]. However, current trends demonstrate that in some instances MSCs remain

72 undifferentiated at the lesion site or in its vicinity, for limited periods of time, or even that

73 only minimal percentages of the MSCs would effectively differentiate and integrate host

74 tissues [14]. Regardless of their differentiation into tissue specific phenotypes, MSCs are

75 often correlated to improved regenerative outcomes [15].

76 These observations were then attributed to the secretion products of those MSCs [16-

77 18] and, in recent years, research has focused on deepening the knowledge on the

78 effective composition of the MSCs secretion [19-22].

79 In most tissues, the key for regenerative efficiency is the re-vascularization of the lesion

80 site and MSCs have been associated with improved angiogenesis in a number of models

81 of disease [23, 24]. As such, MSCs assume a supporting role to the intrinsic mechanisms

82 of tissue regeneration, promoting the re-vascularization processes, providing adequate



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83 perfusion to active healing sites, as well as urging resident regenerative populations to

84 home towards these locations [25].

85 Further, some groups investigated the extent to which the presence of the cells

86 themselves was absolutely essential to the observation of beneficial effects, since

87 regenerative benefit can be observed by the application of MSCs secretion products

88 alone, conventionally designated as the secretome [7, 16-18, 26].

89 The secretome comprises a range of bioactive molecules/factors secreted to the

90 extracellular space, and include soluble proteins, free nucleic acids, lipids and

91 extracellular vesicles. Its composition is particular to individual cells and tissues, and is

92 modulated in response to physiological and/or pathological stimuli [23]. The application

93 of these cell-based products may bring several advantages to the advanced therapies

94 field, namely the decreased cell number requirements and allocated cell storage

95 necessities, ease of tailoring, quality control and dosing, as well as the ready availability

96 for administration in acute scenarios. It is also an evident advantage the application of

97 these bioactive molecules/factors without the presence of MSCs, since it is a safer

98 clinical option considering the risk of neoplastic modifications and the possibility of

99 rejection of the transplanted cells [27], which also facilitates the approval of these

100 treatments by the national and international authorities.

101 Therefore, studies on the composition of the MSCs secretome through metabolic

102 analysis are a valuable tool to the comprehension of the underlying mechanisms to

103 MSCs dynamics and therapeutic effects [28-31].

104 Metabolomic profiling relies mainly on NMR spectroscopy (NMR) and Mass spectrometry

105 (MS) [32-35], yielding information on targeted metabolites’ structure and quantitative

106 distribution [32, 33]. These techniques have been employed on a detailed

107 characterization of fibroblast derived conditioned medium (CM) to investigate their

108 supportive role in human Embryonic Stem Cells [36]. Despite the significant progress



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109 made in the field of structural biology and bio-chemistry, the development and application

110 of these techniques towards the MSCs secretome are still sparse [7].

111 We recently demonstrated the application of proton NMR spectroscopy and

112 implementation of appropriate one (1D) and two (2D) dimensional NMR techniques to

113 the analysis of the metabolic composition of Umbilical Cord Stem/ Stromal Cells (UC-

114 MSCs) conditioned media and changes in the metabolic profile of the culture media in

115 the process of conditioning. These changes were found to be affected mainly by the type

116 of culture media and the duration of the conditioning [20].

117 Alongside the metabolite content, a wide range of growth factors, cytokines, chemokines

118 and extracellular matrix components have already been identified in the CM obtained

119 from differently sourced MSCs [37, 38], and many of them are known to impact on most

120 tissues structure, function and regeneration [24, 39, 40]. Beyond modulating their

121 surrounding environment MSCs are sensitive themselves to signaling factors, altering

122 their secretory profile in response to, as an example, the presence of inflammatory

123 cytokines. This evidence may contribute to the understanding of the eventual difference

124 in the tissue response to cells or to their secretome alone [41], and to the development

125 of strategies to manipulate secretion profiles towards specific needs.

126 In a recent study, we determined that UC-MSCs CM becomes rich in a range of

127 proliferative and anti-apoptotic factors, particularly in transforming growth factor beta 1

128 (TGF-β1), epidermal growth factor (EGF), granulocyte and granulocyte-macrophage

129 colony-stimulating factor (G-CSF and GM-CSF), platelet derived growth factor (PDGF-

130 AA) and vascular endothelial growth factor (VEGF). Also, several other chemokines such

131 as monocyte chemotactic protein-1 e 3 (MCP-1 and MCP-3), chemokine (C-C motif)

132 ligand 5 (CCL5 or RANTES), GRO, and interleukin 8 (IL-8) were observed in increasing

133 levels [20]. This previous analysis enabled the primary definition of UC-MSCs CM

134 composition [20], which we herein expand and compare to Dental Pulp Stem/ Stromal

135 Cells (DPSCs) CM profile.



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136 The first part of the present study focuses on a comparative analysis of the metabolomic

137 and bioactive factors secretion profile of UC-MSCs and DPSCs secretome, through the

138 conditioning process. This aimed to characterize the metabolite profile of the collected

139 CM and to enlighten on the metabolic pathways presiding to the process. Further, the

140 bioactive factors content of the CM was analyzed, focusing on a series of growth factors,

141 cytokines and chemokines, aiming at the comparison of the regenerative potential of the

142 two MSCs population. Finally, the effects of the characterized CMs were addressed on

143 in vitro and in vivo models of angiogenesis.

144

145 1. Materials and Methods

146 1.1. Human Mesenchymal Stem/ Stromal Cells characterization and

147 conditioning protocol

148 Umbilical Cord -MSCs [PromoCellTM (C-12971; Lot No. 1112304.2)] and DPSCs

149 [AllCells, LLC (DP0037F, Lot No. DPSC090411-01)] were cultured with MEM-α,

150 GlutaMAXTM (32561-029, Gibco®) supplemented with MSCs certified FBS (04-400-1A,

151 Biological Industries Israel Beit-Haemek Ltd)(10% v/v), penicillin (100 U/mL)-

152 streptomycin (100 μg/mL)(151140-122, Gibco®), and amphotericin B (2.5 μg/mL)(15290-

153 026, Gibco®). Both cell types were maintained at 37 °C in a 5% CO2 humidified

154 atmosphere. Passage 4 cells were seeded in 75 cm3 T-flasks until confluence was

155 reached. Detachment of confluent cells was achieved by a 5 minutes incubation in 0,05%

156 Trypsin-EDTA (Trypsin-EDTA 0.25%, 25200-072, Gibco®). Cells were re-suspended and

157 counted using 0.4% Trypan Blue (T8154-20ML, Sigma-Aldrich®) and the Countess II FL

158 Automated Cell Counter (InvitrogenTM).

159

160 1.1.1. Mesenchymal Stem/ Stromal Cells phenotype identity

161 The surface marker profiles of UC-MSCs and DPSCs were confirmed though Flow

162 Cytometry, with anti-positive (CD90, CD105, CD44) and negative marker (CD34, CD11b,

163 CD19, CD45, MHC class II) antibodies (Human MSC Analysis Kit, 562245, BD



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164 Biosciences), as described in detail in [42]. Data was acquired using BD FACSCalibur™

165 3 CA Becton Dickinson, BD Biosciences, and data was processed using FlowJo Engine

166 X10.4 (v3.05478, LLC).

167

168 1.1.2. Reverse transcriptase Polymerase chain reaction

169 Reverse transcriptase Polymerase chain reaction (RT-PCR) and qPCR targeting specific

170 genes expressed by pluripotent stem cells was performed. Gene DNA sequences were

171 downloaded from GenBank (www.ncbi.nlm.nih.gov/genbank) and aligned using the

172 Clustal Omega bioinformatic tool from EMBL-EBI

173 (http://www.ebi.ac.uk/Tools/msa/clustalo). The primers sequences are listed in Table 1.

174

175 Table 1. List of primers used, target gene and size of the PCR product.

Primer GenBank target gene PCR product

Pluripotent Stem Cells Markers genes

Fwd: 5’-CCTAAAAGGGCAGAAGAAGGAC-3’ALP humanRev: 5’-TCCACCTAGGATCACGTCAATG-3’

NM_001632.4 444 bp

Fwd: 5’-AACGCTCGACTACCTGTGAA-3'c-Kit human Rev: 5’-GACAGAATTGATCCGCACAG-3’

NM_000222 401 bp

Fwd: 5’-CTTCCTCCATGGATCTGCTTATTC-3’Nanog human Rev: 5’-AGGTCTTCACCTGTTTGTAGCTGAG-3’

XM_011520851.1 265 bp

Fwd: 5’-GAAGCTGGAGAAGGAGAAGCT-3’Oct4 human Rev: 5’-CAAGGGCCGCAGCTTACACAT-3’

NM_002701.5 243 bp

Housekeeping genes

Fwd: 5’-GGCACCCAGCACAATGAAGA-3’β-actin humanRev: 5’-CTGGAAGGTGGACAGCGAGGC-3’

NM_001101.3 100 bp

Fwd: 5’-AGCCGCATCTTCTTTTGCGTC-3’GAPDH humanRev: 5’-TCATATTTGGCAGGTTTTTCT-3’

NM_002046.5 815 bp

176

177 Cultures were harvested and pelleted for total RNA extraction (High Pure RNA Isolation

178 kit (RocheTM)). DNA traces were eliminated with DNase I, and RNA quantity and quality

179 assessed by Nanodrop ND-1000 Spectrophotometer, reading from 220 nm to 350 nm,

180 and stored at -80°C. After, cDNA was synthesized from the purified RNA (kit Ready-To-

181 Go You-Prime First-Strand Beads (GE Healthcare®)).The synthesized cDNA,



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182 corresponding to the mRNA present in the sample, was run for the expression of six

183 genes: two housekeeping genes (β-actin and GAPDH) and four genes used as

184 pluripotent stem cells markers (c-kit, Oct-4, Nanog and ALP). Quantitative PCR (qPCR)

185 was performed in a CFX96TM (BioRad®) apparatus using the iQTM SYBR® Green

186 Supermix (BioRad®). Each pair of primers targeting a gene was used to analyze its

187 expression in the UC-MSCs and DPSCs cDNA, in duplicate, along with a negative

188 control, through defined temperature cycles [95°C for 4 minutes, 35 cycles comprising

189 95°C for 20 seconds, 55°C for 20 seconds and 72°C for 30 seconds ending with Real-

190 Time acquisition, and final extension of 72°C for 7 minutes], and the number of cycle

191 threshold for each well were recorded. The plate containing the amplified genes or qPCR

192 products was kept in ice and observed in a 2% agarose gel (NuSieve® 3:1 Agarose

193 (Lonza)) to check and reinforce the identity of the amplicons, in horizontal

194 electrophoresis apparatus. Under 120 V potential difference for 40 minutes to separate

195 the amplicons. Gel was then observed under UV light and pictures recorded using the

196 GelDoc® 2000 (BioRad®) and Quantity One® software (BioRad®).

197

198 1.1.3. Multilineage differentiation

199 Multilineage differentiation of the UC-MSCs and DPSCs was induced towards

200 Osteogenic, Adipogenic and Chondrogenic phenotypes using specific differentiation

201 media. Differentiation efficiency was assessed by Von Kossa/ Alizarin Red S, Oil Red O

202 and Alcian Blue/ Sulfated Glycosaminoglycans (GAGs) quantification, as detailed in [42].

203

204 1.2. Conditioned Medium collection and analysis

205 For the production of CM, both cellular populations were plated at 6000 cells/ cm2, in

206 triplicates, and cultured in standard culture medium (αMEM 10% FBS) until

207 approximately 80% confluence was reached. Then, the cultures were washed to remove

208 any trace of FBS and other supplements and placed in plain DMEM/F12 GlutaMAX™

209 (10565018, Gibco®) for 24 and 48 hours. Conditioned media samples were collected at



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210 both time-points, centrifuged 1800 xg for 10 min to remove any cellular debris and

211 preserved at -80°C until analysis.

212

213 1.2.1. Metabolomic analysis

214 Proton NMR spectroscopy was used to identify and quantify metabolites in

215 plain/unconditioned and conditioned media collected at two time-points (24 and 48

216 hours) of UC-MSCs and DPSCs in proliferation. A total 600 µL aliquot of each sample

217 was placed into 5 mm NMR tubes and 50 µL deuterium oxide (D2O) containing 0.05 mM

218 sodium trimethylsilyl-[2,2,3,3-d4]-propionate (TSP) was added as a chemical shift and

219 quantitation standard. All NMR spectra were recorded at 300K on a Bruker Avance III

220 600 HD spectrometer, equipped with CryoProbe Prodigy. 1H NMR spectra with water

221 suppression using a 1D NOESY (noesygppr1d) and a Carr-Purcell-Meiboom-Gill

222 (CPMG, cpmgpr1d) pulse sequences [43, 44] were acquired with spectral width of 10000

223 Hz, 32 K data points and 32 scans. The 1D NOESY spectra were collected using 5 s

224 relaxation delay and mixing time of 100 ms. The CPMG experiments (cpmgpr1d) were

225 acquired with relaxation delay 4 s, spin-echo delay between 0.4 and 0.6 ms, and a loop

226 for T2 filter of 20 was used. All free induction decays (FIDs) were processed by 0.3 Hz

227 line broadening and zero filling to 64 K, manually phased, and baseline corrected. Two

228 dimensional 1H/1H COSY and TOCSY spectra were recorded in phase sensitive mode

229 and with water suppression; a relaxation delay of 2 s, 16 or 32 scans, a total 2K data

230 points in F2 and 256 or 512 data points in F1 over a spectral width of 10000 Hz. Two

231 dimensional 1H/13C heteronuclear single quantum coherence (HSQC) experiments

232 were carried out with a spectral width of 10000 Hz for 1H and 27000 Hz for 13C,

233 relaxation delay 1,5 s, Fourier transform (FT) size 2K × 1K. The quantitative distribution

234 of NMR-detectible metabolites in the samples was determined from the integral intensity

235 of characteristic signals in 1H NMR spectra of the samples referenced to the integral

236 intensity of TSP signal, considering the number of the contributing nuclei for that

237 particular resonance signal [45].



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238

239 1.2.2. Bioactive Factors detection and quantification

240 Multiplexing LASER Bead Analysis was performed by Eve Technologies (Calgary,

241 Alberta, Canada) using the Bio-Plex™ 200 system (Bio-Rad Laboratories, Inc., Hercules,

242 CA, USA), similarly to previously described in [46]. The multiplexing technology is based

243 on color-coded polystyrene beads with unique color / fluorophore signatures, and a dual-

244 laser system and a flow-cytometry system. One laser activates the fluorescent dye within

245 the beads which identifies the specific analyte, the second laser excites the fluorescent

246 conjugate (streptavidin-phycoerythrin), and the amount of the conjugate detected by the

247 analyzer is in direct proportion to the amount of the target analyte. The results are

248 quantified according to a standard curve.

249 A broad panel of 57 bioactive factor was assayed [Discovery Assay® TGFβ 3-Plex

250 Cytokine Array (Eve Technologies Corp); Milliplex Human Angiogenesis / Growth Factor

251 kit Discovery Assay® and the Human Cytokine Array / Chemokine Array (Millipore, St.

252 Charles, MO, USA)], including angiopoietin-2 (Ang), bone morphogenetic protein 9

253 (BMP-9), EGF, endoglin (ENG), endothelin-1 (EDN1), eotaxin-1, fibroblast growth factor

254 1 and 2 (FGF-1 and -2), fms-related tyrosine kinase 3 ligand (Flt-3L), follistatin (Fst),

255 fractalkine, G-CSF, GM-CSF, GRO(pan), heparin-binding-EGF (HB-EGF), hepatocyte

256 growth factor (HGF), interferon-alpha 2 (IFNα2), interferon-gama (IFNγ), several

257 interleukins (IL-1α, IL-1β, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12

258 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, IL-18), interferon gama-induced protein 10 (IP-

259 10), leptin, MCP-1, MCP-3, macrophage-derived chemokine (MDC), macrophage

260 inflammatory protein-1 alpha (MIP-1α), macrophage inflammatory protein-1 beta (MIP-

261 1β), platelet-derived growth factor-AA (PDGF-AA) and -AB/BB (PDGF-AB/BB), placental

262 growth factor (PLGF), RANTES/ CCL5, soluble CD40 ligand (sCD40L), transforming

263 growth factor alpha (TGFα), transforming growth beta 1, 2 and 3 (TGF-β 1, -2, and -3),

264 tumor necrosis factor alpha (TNFα), tumor necrosis factor beta (TNFβ), VEGF-A, -C, and

265 -D.



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266 1.3. Conditioned Medium concentration protocol

267 Conditioned Medium obtained from the UC-MSCs and DPSCs was concentrated 5 times

268 (5x), using Pierce™ Protein Concentrator, 3k MWCO, 5-20 mL (88525, Thermo

269 Scientific). Tubes were sterilized as per manufacturer’s instructions, through immersion

270 in 70% (v/v) ethanol for 30 minutes, followed by 2 cycles of 70% ethanol and DPBS

271 (14190144, Gibco®) centrifugations at 3000 xg, for 10 minutes each). Tubes were air

272 dried for 10 minutes and CM placed ate the top compartment. Samples were centrifuged

273 until 5x concentration of sample volume in the upper compartment was achieved.

274 1.4. Human Umbilical Vein Endothelial Cells expansion and maintenance

275 Human Umbilical Vein Endothelial Cells (UVECs) were obtained from Sigma® (200P-

276 05N; Lot No. 3257, Cell Applications, Inc) and expanded using specific expansion

277 medium (211-500, Cell Applications, Inc). Cells were maintained at 37°C and 95%

278 humidified atmosphere with 5% CO2 environment. Passage 4-5 cultures were utilized in

279 the presented assays.

280 1.5. Cell Viability assay

281 Cells were seeded in 24 well plate at 6000 cells/cm2. The cells were allowed to adhere

282 in UVECs expansion medium, overnight. After this period, culture media was replaced

283 by CM supplemented culture media [composed of 80% UVECs expansion medium and

284 20% 5x concentrated UC-MSCs’ CM or DPSCs’ CM; control medium was composed of

285 80% UVECs expansion media and 20% DPBS].

286 At every time point (0, 24, 48, 72 and 96 hours), the culture media was removed and

287 fresh adequate culture media was added to each well, with 10% (v/v) of 10x PrestoBlue®

288 cell viability reagent (A13262, Thermo Fisher Scientific, Molecular Probes, Invitrogen),

289 and UVECs were incubated for 1 hour at 37°C, 5% CO2. The absorbance of the cell

290 culture supernatant was read at 570 nm and 595 nm in a Thermo Scientific Multiskan FC



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291 plate reader, to assess for changes in cell viability. Absorbance readings were

292 normalized, and data corrected to unseeded control wells’ readings.

293 1.6. Senescence and Apoptosis assays

294 To assess for signs of senescence in the cellular populations, β-Galactosidase activity

295 was assayed. Cells were plated in 96-well plates, similarly as described for the cell

296 viability assessment. After 48 hours in CM-supplemented and control media, culture

297 media was removed, and cells were washed once with DPBS and incubated with β-

298 Galactosidase Assay Reagent (75705, Thermo Fisher Scientific), for 30 minutes at 37°C.

299 Absorbance was read at 405 nm.

300 Cultured UVECs were further assessed for apoptosis events, through the detection of

301 Annexin V and Propidium Iodine (PI) staining (BMS500FI, eBioscience). For such, cells

302 cultured for 2 days in each condition were harvested using Trypsin-EDTA, counted and

303 resuspended in provided Binding Buffer. Cells were incubated with Annexin V-FITC and

304 PI, as per manufacturer’s protocol, and FACS analysis was performed using a Coulter

305 Epics XL Flow Cytometer (Beckman Coulter Inc., Miami, FL, USA). Flow cytometry data

306 was processed using FlowJo Engine X10.4 (v3.05478, LLC).

307 1.7. Migration assays

308 Human UVECs, UC-MSCs and DPSCs were plated at 10000 cells/cm2 in a 12 well-plate.

309 Cells were further allowed to expand up to 90% confluency and the centre of the well

310 scratched using a P200 sterile pipette tip.

311 Wells were individually photographed, and standard culture medium was replaced by

312 defined experimental conditions and appropriate controls [UVECs assay - CM media

313 80% UVECs expansion medium and 20% 5x concentrated UC-MSCs’ CM or DPSCs’

314 CM; Control Medium: 80% UVECs expansion media and 20% DPBS; Complete Medium:

315 100% UVECs expansion media; MSCs assay - CM media 80% αMEM 10% FBS and



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316 20% 5x concentrated UC-MSCs’ CM or DPSCs’ CM; Control Medium: 80% αMEM 10%

317 FBS and 20% DPBS; Complete Medium: 100% αMEM 10% FBS].

318 Cells were cultured in the conditions above described and observed for migration and

319 proliferation into the scratched area. Photographs were taken at 0, 10, 14 and 16 hours

320 (for UVECs) and 0, 6 and 24 hours (for MSCs) after experimental media addition and

321 media was changed after each procedure. Photographs were obtained from marked

322 areas along each scratch line, allowing for the monitoring of cell response in multiple

323 areas (Axiovert 40 CFL, Zeiss®).

324 Photographs were then analysed using the ImageJ Software (ImageJ 1.51k, NIH, USA)

325 and the scratched area was measured in time sequenced images, using the ‘MRI Wound

326 Healing Tool’ (http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Wound_Healing_Tool).

327 Six measurements per condition were considered and the results were presented as

328 ‘percentage decrease in scratched area’.

329 1.8. In vitro endothelial tube formation assay

330 For the evaluation of the effect of the CMs on the in vitro endothelial tube formation

331 capacity of the UVECs, cells were plated on Matrigel® (Growth Factor Reduced

332 Matrigel®, Cat. 354230, Corning®) coated 96-well culture plates. A final density of 16000

333 cells per well were plated and specific media added to each group. Endothelial tubes

334 formation was accompanied for up to 12 hours, and photographic record at 40x

335 magnification was obtained at this timepoint (Axiovert 40 CFL, Zeiss®). Recorded images

336 were then analysed using the ImageJ Software (ImageJ 1.51k, NIH, USA).

337 Recorded measurements concerned covered area (%), branching points (#), total tubes

338 (#) and total tube length (px), total loops (#), total loop area (px2) and total loop perimeter

339 (px).



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340 1.9. In vivo vascularization assay

341 For the in vivo assessment, adult male Sasco Sprague Dawley rats [300-350g body

342 weight (b.w.)] were selected. Experimental animals were housed in environmentally

343 controlled facilities, under 12h light-dark cycles. Standard rodent chow and water were

344 provided ad libitum. Normal cage activities were allowed, under standard laboratory

345 conditions. All experimental procedures were approved by the Organism Responsible

346 for Animal Welfare (ORBEA) of the Abel Salazar Institute for Biomedical Sciences

347 (ICBAS), University of Porto (UP) (project 165/2016) and by the Veterinary Authorities of

348 Portugal (DGAV) (project DGAV: 2018-07-11 014510), complying with Directive

349 2010/63/EU of the European Parliament and the European Communities Council

350 Directive 86/609/EEC. Humane Endpoints were considered as recommended by the

351 OECD Guidance Document on the Recognition, Assessment and Use of Clinical Signs

352 as Humane Endpoints for Experimental Animals Used in Safety Evaluation (2000).

353 Before surgical procedure, animals were maintained for two weeks under normal routine,

354 for acclimation.

355 Anaesthesia protocol consisted on intraperitoneal (IP) injection of xylazine-ketamine

356 mixture (Rompun® 20 mg/mL, Bayer®, 12 mg/kg b.w., and Imalgene 1000®, Merial®, 100

357 mg/kg b.w.). The dorsum of each animal was clipped, and the skin asepsis performed

358 with iodopovidone 10% solution (Betadine®).

359 Matrigel® was thoroughly mixed with the Control, Complete and Conditioned Media (80%

360 Matrigel® and 20% of the respective medium). The formulation was maintained in ice, to

361 prevent Matrigel® gelation. The dorsum was virtually divided into four quadrants (each

362 assigned to an experimental group) and a final volume of 500 μL was injected

363 subcutaneously. Upon injection, the formation of a protruding plug was confirmed.

364 Animals were recovered from anaesthesia. After 7 days, animals were placed again

365 under general anaesthesia and sacrificed by lethal intraperitoneal injection of 5% sodium

366 pentobarbital (Eutasil® 200 mg/mL, Ceva).



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367 Injection sites were confirmed and dorsal subcutaneous tissue enclosing the

368 vascularised plug were collected and preserved in paraformaldehyde (3.7-4% buffered

369 to pH 7, Panreac AppliChem®).

370 Collected tissues were processed for routine histopathologic analysis. Sequential

371 sections (3 μm) were prepared and stained with haematoxylin-eosin (H&E) and

372 assessed for the presence and pattern of endotheliocyte/ capillary penetration into the

373 matrix plug.

374

375 1.10. Statistical analysis

376 Data was plotted and treated using GraphPad Prism version 6.00 (GraphPad Software,

377 La Jolla California USA). Data was further analyzed for significant differences. Multiple

378 comparison tests were performed by one-way ANOVA supplemented with Tukey’s HSD

379 post-hoc test. Differences were considered statistically significant at P < 0.05.

380 Significance of the results is indicated according to P values with one, two, three or four

381 of the symbols (*) corresponding to 0.01≤P<0.05; 0.001≤P< 0.01; 0.0001≤P<0.001 and

382 P<0.0001, respectively.

383

384 2. Results

385 2.1. Mesenchymal Stem/ Stromal Cells characterization and conditioning

386 protocol

387

388 Mesenchymal Stem/ Stromal Cells populations depicted characteristic phenotypical

389 markers (Fig 1), presenting ≥ 92% positive population for CD90, CD105 and CD44, and

390 ≤ 2% negative marking for CD34, CD11b, CD19, CD45 and MHC II. Exception was noted

391 for DPSCs population with regard to CD90, which marked positive for only 87-91% of

392 assessed population (Positive population: UC-MSCs - CD90+: 99.23 ± 0.12; CD105+:

393 99.07 ± 0.09; CD44+: 95.70 ± 1.65; NEG+*: 0.13 ± 0.08; DPSCs: CD90+: 90.50 ± 0.35;



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394 CD105+: 99.30 ± 0.06; CD44+: 99.80 ± 0.00; NEG+*: 0.13 ± 0.04; *NEG+ cocktail

395 corresponds to anti-CD34; -CD11b; -CD19; -CD45; and -MHC II antibodies).

396

397 [Fig 1]

398 Fig 1. Surface marker expression for MSCs’ identity of UC-MSCs and DPSCs (positivity

399 for CD90, CD105 and CD44, negativity for a cocktail including CD34, CD11b, CD19,

400 CD45 and MHC II), assessed by Flow cytometry.

401

402 RNA was purified from both UC-MSCs and DPSCs and converted to cDNA using

403 adequate procedures. Primers targeting two housekeeping genes (β-actin and GAPDH)

404 and four typical pluripotency markers (c-kit, Oct-4, Nanog and ALP) were used to support

405 its identity. Fig 2 illustrates the agarose gel of the PCR products and Table 2 details on

406 the average of Threshold cycle (Ct) values. Total RNA was successfully purified from

407 UC-MSCs and DPSCs using the High Pure RNA Isolation kit (Roche), obtaining a

408 concentration of 883.2 ng/µl from UC-MSCs and 158.7 ng/µl from DPSCs. Volumes were

409 adjusted to use only 1.5 µg of total RNA to synthetize corresponding cDNA. This cDNA

410 was used as template in the qPCR reaction. The Ct values show a reasonable

411 amplification of the target genes, in both cell types, resulting in an active expression of

412 these genes in both UC-MSCs and DPSCs. The agarose gel confirms the identity of the

413 genes from the observed molecular weight.

414 The strongest expression is observed in the housekeeping genes, while the lowest

415 expression was observed for the Oct-4 sequence (as evidenced agarose gel profiles (Fig

416 2) and by the lowers and higher ΔCt values determined for the amplification cycles,

417 respectively) (Table 2).

418

419 [Fig 2]

420 Fig 2. Agarose gel profile of the amplification of the selected genes from UC-MSCs (UC)

421 and DPSCs (DP), and a 50 bp DNA ladder reference (50bp).



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422

423 Table 2. Average of Delta Threshold cycle (Ct) values of the amplification of the selected

424 genes from UC-MSCs and DPSCs.

ΔCt valueGene

UC-MSCs DPSCs

Pluripotent Stem Cells Markers genes

ALP 6.03 ± 0.03 6.36 ± 0.01

c-kit 1.96 ± 0.01 1.49 ± 0.02

Nanog 6.41 ± 0.06 6.15 ± 0.02

Oct-4 7.95 ± 0.00 7.26 ± 0.01

425

426

427 Multilineage differentiation: The differentiation capacity of MSCs towards three

428 mesodermal lineages was confirmed (Fig 3). Macroscopic observation revealed

429 mineralized matrix formation, evidenced by Von Kossa staining in both UC-MSCs and

430 DPSCs under osteodifferentiation conditions. Semi-quantitative analysis confirmed

431 observed differentiation, further indicating an increased efficiency and osteogenic

432 propensity of DPSCs when compared to UC-MSCs (2299.31 ± 7.35 μM and 1449.57 ±

433 0.00 μM of ARS, respectively). In adipogenic differentiation, microscopic observation of

434 ORO stained cells evidenced comparable lipid droplets accumulation in differentiated

435 UC-MSCs and DPSCs. Spectrophotometric quantification of ORO staining confirmed

436 observed differentiation and indicate no significant difference between UC-MSCs and

437 DPSCs efficiency. Chondrogenic differentiation groups presented marked light blue

438 staining under macroscopic and microscopic observation, indicating proteoglycan

439 deposition by Alcian Blue staining, confirmed by sGAGs production quantification. UC-

440 MSCs and DPSCs presented no difference in terms of sGAGs production within the

441 differentiated and the undifferentiated wells.

442

443 [Fig 3]



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444 Fig 3. Multilineage differentiation: A) Qualitative evaluation - Osteogenic differentiation:

445 Von Kossa staining after 21 days; Adipogenic differentiation: Oil Red O (ORO) staining

446 after 14 days; Chondrogenic differentiation: Alcian blue staining after 14 days. B) Semi-

447 Quantitative evaluation - Osteogenic differentiation: Alizarin Red S (ARS) concentration

448 (μM) after 21 days; Adipogenic differentiation: Oil Red O (OD570nm) after 14 days; and C)

449 Chondrogenic differentiation: Sulfated GAGs production (μg/ml) after 14 days, assessed

450 by Blyscan™ Glycosaminoglycan Assay (Biocolor, UK). Control: Undifferentiated

451 control; Results presented as Mean ± SEM. a: significantly different from undifferentiated

452 group; b: UC-MSCs differentiated group significantly different from DPSCs differentiated

453 group; c: UC-MSCs undifferentiated group significantly different from DPSCs

454 undifferentiated group (p<0.05).

455

456 2.2. Conditioned Medium collection and analysis

457 Populations from both tissue sources were successfully seeded and reached the desired

458 density within 3 to 4 days. For conditioning time, cells remained well attached and

459 preserved their characteristic spindled shape, although a decrease in the proliferation

460 rate could be empirically observed.

461

462 2.2.1. Metabolomic analysis

463 Proton NMR spectroscopy was used to define the metabolic profiles of UC-MSCs and

464 DPSCs through the analysis of produced CMs. To identify metabolite changes induced

465 by DPSCs and UC-MSCs conditioning, various 1D and 2D NMR experiments were

466 performed, and 1H NMR spectra acquired using appropriate NMR techniques for

467 suppression of the solvent and other interfering signals. The same experimental

468 conditions (temperature, sample/solvent volume, standard (TSP) concentration, NMR

469 acquisition and processing parameters) were used in order to avoid external

470 interferences and to identify particular metabolite variations. The assignment of the

471 proton resonances in NMR spectra and the assessment of the metabolite composition



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472 of the samples were achieved by high resolution 1H NMR spectra analysis, considering

473 specific NMR parameters which reflect structural characteristics of the respective

474 species. The results were verified by the implementation of appropriate 2D NMR (1H/1H

475 COSY, 1H/1H TOCSY, 1H/13C HSQC) spectroscopic techniques and compared to

476 available data in the literature [32, 33]. The assignment of the resonance signals of

477 metabolites in 1H NMR spectra of the samples studied is presented in Table 3.

478 Characteristic resonance signals of metabolites identified in 1H NMR spectra of Plain

479 (unconditioned), UC-MSCs and DPSCs CM are labelled in Fig 4.

480

481 Table 3. 1H NMR chemical shifts and multiplicity of the main metabolites observed in

482 the spectra of Plain, UC-MSCs and DPSCs CM.

483Metabolites Abbrev. Chemical Shifts (ppm) multiplicity

Acetate Ace 1.92(s, CH3)

Alanine Ala 1.48(d, bCH3), 3.78(q, aCH)

Choline Cho 3.19 (s, CH3), 3.51(dd, CH2), 4.06(dd, CH2),

Ethanol Et 1.19 (t, CH3), 3.57 (q, CH2)

Formate For 8.46 (CH)

Glutamate Glu 2.36(t, gCH2), 2.08(m, bCH2), 3.74(dd, aCH)

Glutamine Gln 2.44(t, gCH2), 2.51/2.04(m, bCH2), 3.76(dd, aCH)

Lactate Lac 1.34(d, bCH3), 4.13(q, aCH)

Nicotinamide NA 7.58 (dd, CH), 8.24 (dd, CH), 8.70 (dd, CH), 8.94 (s, CH)

Pyruvate CH3 2.39(s)

Tyrosine Tyr 3.05/3.18 (bCH2), 3.93 (aCH), 7.19(d, H2,6), 6.90(d, H3,5)

α-Glucose 5.24(d, H1), 3.54(dd, H2), 3.72(t, H3), 3.41(t, H4), 3.47(ddd, H5), 3.91/3,72 (dd, H6)

β-Glucose 4.65(d, H1), 3.24(t, H2), 3.49(t, H3), 3.42(t, H4), 3.84(m, H5,6), 3.78(dd, H6)

484

485 [Fig 4]

486 Fig 4. Average 600 MHz 1H NMR spectra (in H2O with 10% D2O) of Plain Medium (A),

487 UC-MSCs CM collected after 24 (B) and 48 hours (C), and of DPSCs CM collected after

488 24 (D) and 48 hours (E). The intensity in the aromatic spectral area (6.5-8.6 ppm)

489 incremented by 10x; the water signal is excluded from the spectra.

490



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491 The visual inspection of the 1H NMR spectra (Fig 4) suggests similar profile shapes but

492 different concentration distribution of the metabolites. To assess characteristic metabolic

493 changes occurring during hMSCs conditioning, comparative quantitative analysis using

494 1H NMR spectra of the samples from the three groups was performed.

495 The metabolites concentration was determined from the integral intensity of

496 characteristic signals in 1H NMR spectra of the samples referenced to the integral

497 intensity of TSP signal used as an internal standard and considering the number of the

498 contributing nuclei for that particular resonance signal. The concentration of the

499 metabolites identified in the 1H NMR spectra of the samples was calculated using the

500 equation [47]:

501

502 Wx =Ix × Nstd × Mx × mstd

Istd × Nx × Mstd

503

504 Where:

505 -Wx represent the mass of the metabolite;

506 -Mx and Mstd are the molar masses of the metabolite and the standard (TSP

507 was used), respectively;

508 -Ix and Istd are the integrated signal area of the metabolite and the standard,

509 respectively;

510 -Nx and Nstd are the number of protons in the integrated signal area of the

511 metabolite and the standard;

512 -mStd represent the mass of the standard.

513

514 The concentration of the metabolites (µM) identified in each group, as well as the

515 chemical shift and assignment of the signal used for quantification are presented in Table

516 4. Statistical significance of encountered differences in Metabolite concentration (μM) is

517 presented in Table S1.



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518 Table 4: Metabolite concentration (µM) calculated from 1H-NMR spectra of unconditioned (Plain Medium), and UC-MSCs and DPSCs conditioned

519 media, after 24 and 48 hours. Data presented in Mean ± SD.

520

521

522Concentration (μM)

Metabolite GroupChem. Shift

(ppm) Plain Medium UC-MSCs 24h UC-MSCs 48h DPSCs 24h DPSCs 48h

Acetate CH3 1.93 2.91 ± 0.02 6.16 ± 0.81 5.15 ± 0.34 2.03 ± 1.17 2.00 ± 0.06

Alanine CH3 1.48 16.34 ± 0.52 120.65 ± 1.76 165.76 ± 2.75 46.42 ± 0.89 141.99 ± 6.17

Choline N(CH3)3 3.21 2.53 ± 0.09 4.60 ± 0.89 3.87 ± 0.17 3.93 ± 0.14 4.20 ± 0.52

Ethanol CH3 1.19 0.84 ± 0.13 8.22 ± 0.18 16.13 ± 0.07 8.47 ± 0.56 21.00 ± 0.30

Formate CH 8.46 0.39 ± 0.19 1.58 ± 0.27 1.92 ± 0.77 1.07 ± 0.09 1.48 ± 0.26

GlutaMAX I (L-Ala) CH3 1.42 94.64 ± 1.24 77.09 ± 0.18 4.97 ± 1.94 88.83 ± 1.77 25.39 ± 9.74

GlutaMAX II (L-Glu) CH2 2.34 153.38 ± 4.29 160.99 ± 4.52 29.72 ± 0.36 160.17 ± 5.22 62.61 ± 8.03

Glutamine CH2 2.45 6.62 ± 0.44 134.98 ± 4.32 175.09 ± 2.54 37.85 ± 0.60 157.38 ± 15.51

Lactate CH 4.12 11.36 ± 0.44 111.44 ± 8.04 131.75 ± 6.58 87.20 ± 1.45 137.52 ± 11.95

Nicotinamide CH 8.94 0.44 ± 0.37 2.55 ± 0.72 2.04 ± 1.77 1.14 ± 0.13 1.16 ± 0.13

Pyruvate CH3 2.37 14.40 ± 0.15 15.67 ± 1.07 7.53 ± 0.37 12.49 ± 4.69 6.97 ± 0.22

Tyrosine 2CH 7.19 21.59 ± 0.26 35.59 ± 1.07 27.51 ± 0.44 26.61 ± 2.11 27.36 ± 1.39

α-Glucose CH 5.24 773.63 ± 1.52 1 112.15 ± 58.57 813.21 ± 16.77 805.17 ± 25.38 860.69 ± 15.68

β-Glucose CH 4.65 945.19 ± 14.00 1 307.92 ± 9.58 1 038.61 ± 63.04 913.16 ± 2.84 998.30 ± 19.05



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523 All 1H NMR spectra are dominated by the proton resonance signals of glucose but

524 several low molecular weight compounds such as amino acids (e.g. alanine, tyrosine),

525 organic acids (e.g. lactate, acetate, citrate, formate) and choline are identified (Fig 4).

526 Additionally, in the spectra of the Plain Medium, resonance signals characteristic for L-

527 alanyl-L-glutamine (GlutaMAXTM I (L-Ala) at 1.42 and GlutaMAXTM II (L-Glu) at 2.37 ppm)

528 dipeptide from GlutaMAXTM are clearly observed. The intensity of these resonances

529 significantly decreases through the cell conditioning process, which is especially evident

530 after 48 hours of culture conditioning. Glutamine and alanine, the resulting compound

531 from GlutaMAXTM and glutamate’s metabolism, expectedly rise through culturing times.

532 Remarkably, UC-MSCs’ CM increase in these metabolites’ content occurs at 24 hours,

533 before significant GlutaMAXTM consumption can be observed. For the DPSCs

534 population, this process appears to occur at a slower rate (Fig 5A and 5B).

535

536 [Fig 5]

537 Fig 5. Dynamics of GlutaMAXTM breakdown to glutamine and alanine of UC-MSCs (A)

538 and DPSCs (B) populations; and Dynamics of Pyruvate catabolism into acetate, lactate

539 and ethanol of UC-MSCs (C) and DPSCs (D) populations.

540

541 Glucose is the most prominent metabolite identified in the spectra and therefore in

542 greatest concentration. In the media collected after 24 hours of conditioning of UC-

543 MSCs, a significant increase of α and β-Glucose content is observed, when compared

544 to the Plain Medium (which was supplied to the culture at the start of the conditioning

545 period), as well as when compared to the DPSCs at the same conditioning time. This

546 initial spike in glucose content decreases in the following hours, reaching back to values

547 not significantly different from the Plain Medium. Human DPSCs present no significant

548 variation in glucose content throughout the study period.

549 Pyruvate is an energetic compound that is catabolised through aerobic or anaerobic

550 pathways. The analysis of the Plan Media confirms it is one of the supplemented



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551 metabolites. Both cell types appear to present a tendency for pyruvate consumption in

552 the 48 hours of conditioning (Fig 5C and 5D), but significance in such reduction is only

553 found for UC-MSCs. Catabolites resulting from its metabolism are also detected in the

554 spectra, such as acetate, lactate and ethanol. Acetate accumulated in UC-MSCs

555 cultures, particularly in the first 24h (Fig 5C). In DPSCs CM, Acetate concentrations

556 remained stable though the evaluation (Fig 5D). Lactate, on the other hand, accumulated

557 strongly within the first 24 hours in both cases, with little increase afterwards, reaching

558 maximum concentration at 48 hours of incubation. Ethanol presented a steady rate of

559 accumulation in the culture supernatant for both UC-MSCs and DPSCs.

560 Other compounds such as choline, formate, tyrosine and nicotinamide are also detected

561 at low levels in both Plain and Conditioned media.

562

563 2.2.3. Bioactive Factors detection and quantification

564 Multiplexing LASER Bead Analysis was performed for the detection of a total of 57

565 Bioactive factors in the CM obtained from UC-MSCs and DPSCs, after 24 and 48 hours

566 of conditioning. Obtained measurements were normalized to the Unconditioned/ Plain

567 medium (naturally devoid of artificial bioactive factors), run as blank sample. Fluorescent

568 readings were plotted against a standard curve and calculated concentrations are

569 presented in Table 4. Statistical significance of encountered differences in the detected

570 Bioactive factors (pg/mL) is presented in Table S2.

571

572 Table 4. Bioactive factors detection and quantification (pg/mL) calculated from

573 Multiplexing LASER Bead Analysis of UC-MSCs and DPSCs CM after 24 and 48 hours

574 of conditioning. Data presented in Mean ± SEM. Bioactive factors presenting no to

575 minimal signal intensity in grey. ND, not detected.

576

Concentration (pg/mL)Bioactive Factor

UC-MSCs 24h UC-MSCs 48h DPSCs 24h DPSCs 48h



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Angiop-2 ND 10.66 ± 0.45 ND 1.86 ± 1.00BMP-9 ND 0.22 ± 0.02 ND 0.00 ± 0.00EGF 0.23 ± 0.13 0.07 ± 0.04 0.28 ± 0.02 0.68 ± 0.10

Endoglin ND 47.74 ± 3.25 0.93 ± 0.93 2.56 ± 0.39Endothelin-1 ND 1.21 ± 0.07 ND 0.03 ± 0.03

Eotaxin-1 ND 1.50 ± 0.49 4.37 ± 2.08 3.09 ± 1.54FGF-1 ND 1.97 ± 0.63 ND 0.26 ± 0.22FGF-2 ND 212.27 ± 12.39 ND 7.06 ± 7.06Flt-3L ND 1.37 ± 0.69 ND 0.76 ± 0.22

Follistatin 143.45 ± 10.69 1101.53 ± 293.83 202.04 ± 29.56 1309.40 ± 110.60Fractalkine 3.37 ± 0.95 23.76 ± 13.03 ND ND

G-CSF ND 1041.30 ± 113.67 ND 0.49 ± 0.25GM-CSF 1.24 ± 0.34 2.45 ± 1.07 ND 1.04 ± 0.25GRO pan 17.80 ± 7.49 966.84 ± 177.44 ND 12.19 ± 1.45HB-EGF 0.02 ± 0.00 0.97 ± 0.11 0.04 ± 0.04 0.10 ± 0.03

HGF 113.99 ± 7.25 4436.31 ± 516.74 0.37 ± 0.37 69.86 ± 18.50IFNα2 3.19 ± 0.99 3.04 ± 0.41 10.95 ± 5.08 2.28 ± 1.11IFNγ 0.27 ± 0.05 1.22 ± 0.11 0.40 ± 0.16 NDIL-10 ND 0.29 ± 0.05 ND ND

IL-12(p40) 2.80 ± 0.89 3.49 ± 0.63 0.64 ± 0.11 NDIL-12(p70) 0.32 ± 0.17 0.40 ± 0.04 0.38 ± 0.03 0.41 ± 0.14

IL-13 1.02 ± 0.06 0.76 ± 0.12 0.27 ± 0.04 1.15 ± 0.13IL-15 0.65 ± 0.02 0.80 ± 0.19 0.37 ± 0.04 0.49 ± 0.03

IL-17A ND ND ND NDIL-18 ND ND ND NDIL-1B 0.56 ± 0.08 0.35 ± 0.05 0.50 ± 0.26 0.43 ± 0.17

IL-1RA 0.54 ± 0.14 0.78 ± 0.27 ND NDIL-1α 4.45 ± 0.31 7.10 ± 0.73 ND NDIL-2 ND ND ND NDIL-3 0.38 ± 0.21 ND ND NDIL-4 0.81 ± 0.35 ND ND NDIL-5 ND ND 0.01 ± 0.00 0.05 ± 0.03IL-6 31.24 ± 2.47 470.56 ± 33.61 ND NDIL-7 0.12 ± 0.05 0.65 ± 0.10 ND NDIL-8 48.18 ± 2.89 1863.05 ± 10.77 0.02 ± 0.01 1.23 ± 0.14IL-9 ND 0.30 ± 0.06 ND 0.38 ± 0.29

IP-10 1.90 ± 0.10 ND ND 1.23 ± 0.10Leptin 1.76 ± 0.00 216.56 ± 17.66 39.62 ± 24.47 12.09 ± 12.09MCP-1 972.36 ± 83.42 2626.08 ± 236.18 N.D. 0.44 ± 0.33MCP-3 6.84 ± 3.45 53.53 ± 6.48 2.43 ± 0.89 7.23 ± 2.77MDC ND 32,13 ± 6,16 ND ND

MIP-1α ND ND ND NDMIP-1β ND ND ND ND

PDGF-AA 0.14 ± 0.01 0.56 ± 0.04 ND 0,02 ± 0,01PDGF-BB 2.06 ± 0.56 3.15 ± 1.35 2.24 ± 0.67 4.29 ± 1.97

PLGF 0.17 ± 0.04 0.11 ± 0.03 0.13 ± 0.06 4.35 ± 0.30RANTES 12.35 ± 2.32 73.61 ± 5.56 4.52 ± 1.58 1.53 ± 1.12



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sCD4L ND ND ND NDTGF-α ND ND 0.26 ± 0.08 NDTGF-β1 11.96 ± 2.18 582.49 ± 56.37 4.08 ± 2.08 53.01 ± 23.04TGF-β2 0.69 ± 0.15 174.20 ± 17.52 0.13 ± 0.13 29.33 ± 5.09TGF-β3 0.09 ± 0.09 1.77 ± 1.25 ND 0.20 ± 0.16TNFα 0.05 ± 0.01 0.24 ± 0.07 ND 0.08 ± 0.00TNFβ ND 0.86 ± 0.17 ND 1.38 ± 0.27

VEGF-A 17.25 ± 3.48 1.34 ± 0.28 14.31 ± 8.75 2041.55 ± 45.66VEGF-C 1.90 ± 0.13 486.48 ± 23.48 ND 30.71 ± 1.67VEGF-D 0.57 ± 0.22 1.90 ± 0.48 0.52 ± 0.17 ND

577

578

579 After 24 hours of conditioning most factors presented no to little expression. Some of the

580 factors gained expression after 48 hours, becoming detectable and, occasionally, in

581 significant concentrations. Overall, none to minimal signal intensity was detected in 36

582 of the 57 assayed factors, combining the two timepoints.

583 A similar pattern of superior secretion by UC-MSCs is observed throughout, highlighting

584 HGF, IL-8, and MCP-1 expressive quantities. Exception is appointed to follistatin and,

585 most significantly, to VEGF-A, where DPSCs depict increased levels when compared to

586 UC-MSCs.

587

588 2.1. Cell Viability, Senescence and Apoptosis assays

589 The CM obtained from MSCs cultures was employed as culture medium supplement for

590 in vitro culture of UVECs, to assess for its ability to sustain the activity and proliferation

591 of the endothelial populations. PrestoBlue® viability was used as a metabolic indicator.

592 Obtained corrected absorbance values are presented in Fig 6 and Table 5 (statistical

593 differences on observed measurements detailed in Table S3).

594 At 24 hours, UVECs cultured in medium supplemented with DPSCs CM depict slightly

595 increased activity, with no other noteworthy differences. As time progresses (48 and 72

596 hours), both DPCSs and UC-MSCs CM supplemented groups sustain superior cellular

597 activity than Complete and Control media. At about 72 hours of culture, the UVECs

598 cultured in CM supplemented media reached confluence and entered the plateau stage.



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599 The activity of Control medium group also decreased from that point onwards, although

600 cellular confluence was unmatched to the CM supplemented groups. At 96 hours, only

601 the Complete medium continued to present an increase in cellular metabolism but

602 remained slightly inferior to that provided by DPCSs CM.

603

604 [Fig 6]

605 Fig 6. Corrected absorbance readings of Presto Blue® Assay of UVECs, at 0, 24, 48, 72

606 and 96 hours of expansion in control or CMs supplemented media. Values are presented

607 as Mean ± SD (A); Apoptosis (Annexin-V/ PI) assay of UVECs, after 48 hours of

608 expansion in Complete, Control or CMs supplemented media. Results presented as

609 percentage (%) of cells of Viable, Early Apoptotic, Late Apoptotic and Death cells, as

610 Mean ± SD (B).

611

612 Table 5: Corrected absorbance readings of Presto Blue® Assay of UVECs, at 0, 24, 48,

613 72 and 96 hours of expansion in control or CMs supplemented media. Values are

614 presented as Mean ± SD.

615

Corrected Absorbance

Complete Medium

Control UC-MSCs CM DPSCs CM

0 h 0.171 ± 0.012 0.161 ± 0.015 0.165 ± 0.011 0.165 ± 0.008

24 h 0.254 ± 0.056 0.215 ± 0.014 0.284 ± 0.018 0.304 ± 0.014

48 h 0.442 ± 0.014 0.420 ± 0.015 0.477 ± 0.015 0.509 ± 0.007

72 h 0.464 ± 0.015 0.456 ± 0.012 0.546 ± 0.005 0.562 ± 0.003

96 h 0.465 ± 0.014 0.424 ± 0.010 0.459 ± 0.040 0.502 ± 0.019

616

617 Senescent events on the cultured populations were assessed through the β-

618 galactosidade activity. Control medium presented increased enzyme activity when

619 compared do the CM supplemented groups (Corrected absorbance, as mean ± SD –

620 Control: 0.0025±0.0030; UC-MSCs CM 0.0003±0.0004; DPCSs CM: 0.0000±0.0000;

621 0.001≤P< 0.01). CM supplemented groups presented no significant difference from the



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622 UVECs maintained in their standard Complete medium (Corrected absorbance, as mean

623 ± SD: 0.0009±0.0007).

624 As for apoptosis events in the cultured population, none of the CM supplemented groups

625 revealed noteworthy impact in cellular viability (Fig 6B, Table 6 and Table S4). The most

626 striking observation is that the Control group presented increased events of early and

627 late stage apoptosis, significantly decreasing the percentage of viable cells. As for the

628 groups supplemented with DPSCs or UC-MSCs CM, the first presented increased

629 cellular viability, as opposed to the early apoptotic and cell death events identified in the

630 UC-MSCs CM supplemented group. The CM supplemented groups matched the events

631 of each stage recorded for UVECs cultured in their standard Complete media, except for

632 the early apoptosis, where DPSCs CM provided an inferior percentage.

633 Table 6: Apoptosis (Annexin-V/ PI) assay of UVECs, after 48 hours of expansion in

634 Complete, Control or CMs supplemented media. Results presented as percentage

635 (%) of cells of Viable, Early Apoptotic, Late Apoptotic and Dead cells, as Mean ±

636 SD.

637

Apoptosis(Annexin V/ PI)

Complete Medium


Viable Cells

95.07 ± 0.25 89.47 ± 0.41 94.33 ± 0.41 95.17 ± 0.29

Early Apoptosis

2.79 ± 0.06 5.93 ± 0.09 2.71 ± 0.06 2.47 ± 0.05

Late Apoptosis

1.97 ± 0.21 4.43 ± 0.28 2.71 ± 0.28 2.09 ± 0.16

DeadCells

0.17 ± 0.01 0.11 ± 0.02 0.48 ± 0.10 0.27 ± 0.07

638

639

640



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641 2.2. Migration assays

642 After wound induction on the cellular monolayers with the pipette tip, the area of the

643 produced defect did not differ between groups/wells, deeming them adequate for

644 comparison through the healing time.

645 In one instance, we investigated the chemotactic effect of the MSCs CMs on endothelial

646 populations, represented by HUVECs. No significant differences in recovered area of the

647 wound were identified until 14 hours into the assay, when DPSCs CM supplemented

648 groups depicted increased percentage of covered wound area, when compared to the

649 Complete and Control media (0.0001≤P<0.001)(Fig 7 and Table 7). At the final

650 assessment time (16 hours), both CM supplemented groups presented over 90% area

651 coverage, while Complete and Control media groups remained under 85% coverage of

652 the originally induced ‘scratch’ (0.0001≤P<0.001 to P<0.0001).

653

654 [Fig 7]

655 Fig 7. Migration assay of UVECs, at 0, 10, 14 and 16 hours in control or CMs

656 supplemented media. Values are presented as percentage (%) of wound area coverage

657 from the 0 hours baseline, 40x magnification.

658

659 Table 7: Migration assay of UVECs, at 0, 10, 14 and 16 hours in control or CMs

660 supplemented media, wounded area in 105 pixels. Values are presented as Mean ± SD.

661

Area(105 pixels)

Complete Medium


0 h 33.207 ± 2.198 33.378 ± 1.573 35.089 ± 0.718 33.486 ± 2.322

10 h 13.332 ± 1.547 15.436 ± 2.372 14.132 ± 2.694 12.431 ± 3.408

14 h 10.703 ± 3.403 10.490 ± 2.028 7.993 ± 3.641 4.366 ± 2.235

16 h 5.298 ± 1.202 5.132 ± 0.915 3.470 ± 0.295 3.244 ± 0.779

662

663

664 Further, we assessed the effect of the CMs on the chemotaxis of the two MSCs

665 populations under study. After 6 hours of ‘scratching’ and media replacement of the



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666 MSCs monolayers, no differences were observed in cellular migration in Control and

667 Complete Medium groups between, and within the two MSCs sources (Fig 8 and Table

668 8). The CM supplemented groups depicted superior area, with exception for CMs

669 supplemented DPSCs, which did not differ significantly from the same population

670 cultured in αMEM 10% FBS. No differences were patent in UC-MSCS and DPSCs

671 supplemented with either CM (0.0001≤P<0.001 to P<0.0001).

672 At the final assessment of 24 hours, UC-MSCs in CM supplemented media presented

673 increased migration into the area than the un-supplemented Control and αMEM 10%

674 FBS groups. They also presented increased motility than the DPSCs populations

675 (0.0001≤P<0.001 to P<0.0001). No difference in response to the two CMs was

676 observed to within each MSCs type, but UC-MSCs responded with superior migration

677 when paired to the DPSCs (0.0001≤P<0.001 to P<0.0001).

678

679 [Fig 8]

680 Fig 8. Migration assay of hMSCs, at 0, 6 and 24 hours of expansion in control or CMs

681 supplemented media. Values are presented as percentage (%) of wound area coverage

682 from the 0 hours baseline, 40x magnification.

683

684 Table 8: Migration assay of hMSCs, at 0, 6 and 24 hours of expansion in control or CMs

685 supplemented media, wounded area in 105 pixels. Values are presented as Mean ± SD.

686

Area(105 pixels)

Complete Medium Control UC-MSCs CM DPSCs CM

0 h 41.42 ± 2.61 37.55 ± 4.54 35.06 ± 3.98 36.56 ± 5.14

6 h 29.13 ± 3.82 27.80 ± 2.73 19.62 ± 2.98 18.83 ± 2.11

UC

-MSC

s

24 h 8.72 ± 3.45 12.64 ± 2.43 3.17 ± 1.08 2.97 ± 1.97

0 h 42.51 ± 5.87 41.26 ± 3.27 41.42 ± 2.87 42.74 ± 2.05

6 h 25.30 ± 3.71 28.55 ± 3.53 22.34 ± 2.28 21.79 ± 2.75

DPS

Cs

24 h 11.01 ± 3.65 21.34 ± 5.73 12.65 ± 2.54 8.09 ± 2.66



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687 2.3. In vitro endothelial tube formation assay

688 The in vitro angiogenic potential of the CMs was evaluated through the tube formation

689 capacity of UVECs, cultured in Complete, Control, UC-MSCs and DPSCs CM

690 supplemented media. Control group presented the least effective tube formation

691 capacity, while CM supplemented groups provided enhanced in vitro angiogenic

692 potential (Fig 9).

693 It was noticeable that UVECs exposed to UC-MSCs and DPSCs CM supplemented

694 media formed consistently well-defined tubular networks, with evenly distributed and

695 regularly shaped loops in the Matrigel® matrix. Complete medium supplementation

696 resulted in defined tubes, although loop distribution appears less homogeneous than in

697 the CMs groups. Occasional events of incomplete tube connection from the branching

698 points were observed. In the Control group, the dominant observation is the incomplete

699 bridging attempts and short unconnected tubes, resulting in large and hill shaped loops.

700 Regarding the number of branching points, no differences were encountered between

701 groups. In terms of total loops (formed from the connection of the branched tubes) and

702 total tube length, Complete medium and CM supplemented groups showed significative

703 superior performance, when compared to Control medium. Although both CMs provided

704 increased results over the Complete medium group, statistical significance was only

705 noted for the DPSCs CM supplemented group. No significant difference was found

706 between the two CMs. The surface area covered by UVECs reflected the tube formation

707 efficiency observed (Fig 9 and Table 9).

708

709 [Fig 9]

710 Fig 9. Tube formation assay after 12 hours of UVECs exposure to Complete Control,

711 UC-MSCs or DPSCs supplemented media (upper panel). Graphical representation of

712 total branching points, total loops, covered area and total tube length observed in each

713 group (lower panel). Values are presented as Mean ± SD. Significant differences

714 indicated according to P values with one, two, three or four of the symbols (*)



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715 corresponding to 0.01≤P<0.05; 0.001≤P<0.01; 0.0001≤P<0.001 and P<0.0001,

716 respectively.

717

718 Table 9. Tube formation assay after 12 hours of UVECs exposure to Complete, Control,

719 UC-MSCs or DPSCs supplemented media: total branching points, total loops, covered

720 area and total tube length observed in each group. Values are presented as Mean ± SD.

721Tube Formation

AssayComplete medium Control UC-MSCs CM DPSCs CM

Covered area (%) 16.93 ± 2.50 13.75±3.82 18.89±1.98 21.69±1.93

Branching points (#) 23.14 ± 5.52 19.17±4.62 21.09±6.04 23.10±4.51

Total tubes (#) 47.29 ± 12.57 44.67±12.61 43.27±13.91 46.60±9.00

Total tube length (px) 15415.00 ± 582.38 12292.00±1169.33 16522.86±1239.79 17933.00±1858.48

Total loops (#) 10.43 ± 1.81 7.00±2.19 12.91±1.81 14.45±1.97

Total loop area (px2) 503384 ± 476226 374678±331769 335432±229543 279728±170898

Total loop perimeter (px) 3087.11 ± 2047.40 3738.14±2591.60 2833.46±1241.24 2459.92±1054.61

722

723 2.4. In vivo vascularization assay

724 After 7 days of subcutaneous implantation, the Matrigel® plugs were evidenced and

725 collected. Discrete vascular penetration was observed macroscopically in all groups.

726 Microscopically, the Complete Medium and the UC-MSCs CM groups presented

727 apparent increased capillary density than the Control group (Fig 10). In the CM groups,

728 the capillary penetration reached deeper into the matrix and displayed a more mature

729 nature, evidenced by the patent vascular structures containing circulating erythrocytes.

730

731 [Fig 10]

732 Fig 10. In vivo re-vascularization (Matrigel® Plug) assay, depicting capillary

733 penetration into the matrix in the Control, Complete, UC-MSCs or DPSCs CMs

734 groups; haematoxylin-eosin (H&E), 200x magnification.

735



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736 3. Discussion and Conclusions

737 The disclosure of the composition of MSCs populations’ secretome is key to the

738 comprehension of the underlying mechanisms of their therapeutic action. Therefore, it is

739 key to understand the basal profile of secretion of these MSCs populations, to grant basis

740 for the selection of the MSCs systems most suitable for each intended therapeutic

741 application. For such, we conducted an analytical study focusing on the investigation of

742 the metabolomic and bioactive factors composition of the secretome of hMSCs

743 originated from two of the most promising sources for medical applications: the UC-

744 MSCs and the DPSCs. These sources may come to gain ground for MSCs-based

745 therapies due to the non-/ minimally invasive and ethically accepted collection

746 procedures, as well as for the increasingly available private and public banking options

747 worldwide.

748 The international scientific community has long debated the criteria for MSCs character

749 assignment and issued a conciliatory guiding line of features for the definition of

750 populations classified as MSCs [2], considering their phenotypic and differentiation

751 capacities, which was confirmed on the hMSCs herein explored. We observed an

752 increased capacity for DPSCs to differentiate towards mineralising populations, as

753 reported by [48], but did not confirm their decreased capacity for adipogenesis since both

754 UC-MSCs and DPSCs displayed comparable Oil Red O uptake. Further, a panel of

755 specific genes has been reported to correlate with the proliferative capacity of

756 undifferentiated MSCs and pluripotency/ multilineage differentiation capacity. Both UC-

757 MSCs and DPSCs presented comparable expression of ALP, c-kit and Nanog. ALP has

758 been described to relate to the proliferative stage of undifferentiated stem cells of

759 embryonic and mesenchymal origin, while Nanog expression in MSCs is associated with

760 the transition from in vivo quiescence to adaptation to in vitro growth condition. Both

761 genes are described to downregulate as lineage commitment occurs, except for the

762 osteoblastic phenotype conversion, where it is characteristically upregulated [49, 50]. C-



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763 kit is the gene coding for the receptor for the stem cell factor, also observed highly

764 proliferative populations and related to differential lineage differentiation capacity [51].

765 Oct-4 was expressed to a lower extent in both cellular populations. Oct-4, along with

766 Nanog, is associated to MSCs populations plasticity [52]. Other authors report that Oct-

767 4 was not identified in cultured human adult MSCs [50].

768 These populations were therefore expanded in culture and elicited to secrete bioactive

769 molecules to the surrounding media, through the conditioning process. The conditioning

770 process was handled under serum-free conditions, so that the only factors detected in

771 the CM were those produced by the UC-MSCs and DPSCs and not those bared to the

772 system by exogenous supplementation.

773 Addressing the obtained CM from a metabolomic perspective (through 1H-NMR

774 spectroscopy), we identified glucose as the dominant metabolite in the spectra. Nuschke

775 et al. recently investigated the metabolism of glucose in MSCs populations (bone

776 marrow) and determined this to be a major limiting factor for MSCs survival. They

777 describe a steady consumption rate for glucose in semi- and confluent cultures [53], a

778 tendency that we could not observe in the cultured UC-MSCs and DPSCs. Indeed, UC-

779 MSCs present an increase in both α- and β-glucose content, suggesting a possible

780 engagement to gluconeogenesis and the utilization of other bioenergetic substrates for

781 energy production. The observed profiles suggest that, although significant amounts of

782 glucose are provided by plain media, conditioning cells do not solely rely on glycolytic

783 pathways for ATP and NADP production.

784 Pyruvate is therefore suggested as one of the crucial energetic metabolites that enters

785 a variety of metabolic pathways, for the aerobic production of ATP [through its conversion

786 into acetate/acetylCoA and entrance into the citric acid cycle]. Pyruvate is detected at

787 relatively low concentrations, with a small tendency for consumption as conditioning time

788 progresses. Pyruvate is initially provided by the media, but the maintenance of its

789 concentration in the media suggest intrinsic production by the MSCs, through non-



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790 glucose dependent alternative synthesis pathways. Acetate levels, an intermediate

791 product for the aerobic pathway of pyruvate catabolism, also remain fairly constant, as it

792 is not expected to accumulate, but to continue its path through the citric acid cycle

793 towards energy production. The build-up of choline and formate (related to the

794 production of acetate) converge to this assumption.

795 Evidence of anaerobic metabolism is also observed throughout the conditioning period,

796 providing accumulation of ethanol and lactate, both of which derived mainly from

797 fermentation processes of the same energetic precursor pyruvate.

798 The observed accumulation of metabolites related to both bioenergetic processes

799 supports the combination of both aerobic and anaerobic pathways for energy production

800 by conditioning MSCs populations.

801 Other substrates dynamics are worth highlighting. GlutaMAXTM is a formulation whose

802 hydrolysis results in the release of alanine and, ultimately, of L-glutamine, which is an

803 essential nutrient in cell cultures for energy production as well as protein and nucleic acid

804 synthesis [54]. This profile of consumption of GlutaMAXTM and build-up of its by-products

805 is observable in both cellular populations. Little consumption is detected at 24 hours, but

806 the process becomes very significant after 48 hours in culture. Additionally, UC-MSCs

807 seem to entail on this metabolic pathway faster that DPSCs. The metabolic dynamics

808 observed supports the survival and biosynthetic pathway endured by MSCs that,

809 amongst other compounds, results in the production and/or release of bioactive

810 molecules into their surroundings.

811 Through the evaluation of the bioactive factors content of the UC-MSCs and DPSCs

812 secretome, the paracrine function of MSCs in therapeutic setups is substantiated. This

813 topic has been granted greater attention in research when compared to the metabolomic

814 composition of the MSCs secretome, and various cell sources have been researched

815 besides the bone marrow standard [55-63], such as the adipose tissue [64-67], the dental

816 pulp and other dental tissues [61, 68-71], the umbilical cord tissue [63, 72], cord blood

817 [73], and other placental/ foetal tissues [63, 74]. These works report the identification of



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35

818 most of the angiogenic factors herein investigated, such as Ang-2 [63], Endothelin-1 [65,

819 70], FGF-2 [57-59, 63, 64, 69, 73], G-CSF [67], HB-EGF [73], HGF [59, 64, 65, 72, 73],

820 IL-8 [57, 63, 68, 72, 74], PLGF [58] [63], VEGF [55-62, 64, 65, 68-70, 73], and TGF-β

821 [60, 64] [63].

822 We identified a series of pro-proliferative/ anti-apoptotic (TGFβs, FGF-2, G-CSF, HGF

823 and VEGFs) and chemotactic factors (RANTES, GRO, MCP and MDC), as well as

824 relevant cytokines (IL-6 and IL-8). Some other factors are described in the literature,

825 which we were not able to detect in significant amounts, such as GM-CSF [64]. PDGF is

826 another angiogenic factor previously reported to be produced by DPSCs [69] that we

827 were unable to detect in the conditioned media.

828 Exploring into the specific functions of the identified factors, the VEGF family members

829 are primary factors in the MSCs pro-angiogenic character [75, 76]. Interestingly, most

830 authors do not take into account the existence of the different isoforms of VEGF, and

831 rather quantify its presence in bulk. We herein demonstrate that the differential detection

832 of VEGF isoforms is of great importance, since the assayed cellular sources

833 demonstrated clear differences in the isoforms profile content. Human DPSCs were here

834 demonstrated to predominantly secrete VEGF-A (that connects to endogenous re-

835 vascularization response [77]), while UC-MSCs were more prone to VEGF-C secretion

836 (prone to neurogenesis induction, without exerting angiogenic effects [78]), which may

837 impact on their potential for tissue regeneration therapies. Janebodin and colleagues

838 [61] also report source dependent differential isoform secretion, and demonstrated that

839 DPSCs secreted superior levels of VEGF-A than BM-MSCs. They also report the

840 secretion of VEGF-D, and to significantly greater extents than BM-MSCs, while we were

841 unable to detect significant VEGF-D signal, especially after 48 hours of secretion. VEGFs

842 (and Ang) directly stimulate endothelial cells populations to proliferate and migrate, and

843 to organise in tube like vascular networks of increased stability and maturation [55].

844 Other mitogens are described for endothelial populations [79], such as FGF-2 and PLGF,

845 which we failed to observe in relevant amounts [80], in line with the recent reports for



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846 UC-MSCs [63] and for DPSCs [62, 68]. HGF, the dominant bioactive factor identified in

847 the UC-MSCs secretome, is recognised as anti-apoptotic and pro-mitogenic in various

848 tissues [81], and was previously identified in UC-MSCs [76] and adipose derived-MSCs

849 [64]. The strong expression of TGF-βs (particularly TGF-β1) by UC-MSCs could raise

850 flags to their potential effect on the stimulation of fibroblastic population at a lesion site,

851 but the combined secretion with antagonistic molecules, such as HGF and Follistatin

852 may be the key to the antifibrotic character of MSCs populations [82]. IL-8 similarly

853 displays pro-angiogenic features [64], promoting endothelial proliferation, migration and

854 in vitro tube network formation [72].

855 These observations lead to the conclusion that both cell sources successfully supplied

856 endothelial populations with stimulatory factors for their in vitro proliferation, migration

857 and tube formation capacity, and forestalled senescence and apoptosis factors deprived

858 endothelial cells. Indeed, the CM efficiently outweighed the complete endothelial cell

859 expansion media utilised as positive control.

860 IL-8 and G-CSF are potent chemoattractant molecules [83], with recognised role in the

861 homing of endogenous and delivered MSCs to lesion sites [84]. We observed great

862 amounts of MCP-1 secretion by UC-MSCs, but not from DPSCs, colliding with

863 Bronckaers’ observations [68]. Potapova [57] reports BM-MSCs to also secrete MCP-1,

864 in inferior proportion than IL-8 and IL-6. We observed an inverse pattern, since UC-MSCs

865 secreted much expressive concentrations of MCP-1 instead of the ILs. Other entities

866 such as eotaxin, fractalkine, GRO, and MDC and RANTES have been demonstrated to

867 be chemoattract to MSCs [25, 85, 86]. The UC-MSCs maintain increased secretion of

868 the molecules and, similarly to what was observed in the endothelial populations, the

869 action of the CMs in the migration of the MSCs populations did not display equivalent

870 differences. Conditioned media from both UC-MSCs and DPSCs promoted overall

871 increased migration of the MSCs, seldom comparable to the stimuli provided by standard

872 expansion media. The UC-derived populations appear to be more responsive to the CM

873 vehiculated stimuli. Additionally, the UC-MSCs also appear to respond to the bioactive



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874 factors produced by themselves during the migration challenge, as the un-supplemented

875 controls display some degree of in-defect proliferation, exceeding that of DPSCs. The

876 distinct response profile may relate to the expression of chemokine receptors by MSCs,

877 that varies though species, culturing time and, suggestively, though tissue source for

878 isolation [25].

879 Our report on the composition of the CMs is therefore consistent with the properties

880 attributed to MSCs, suggesting that UC-MSCs provide a wider variety and greater

881 concentration of relevant growth factors and cytokines. The proposed difference in the

882 secretory potential of UC-MSCs and DPSCs was explored in vitro and in vivo focusing

883 on their effects on endothelial populations, as well as on the MSCs themselves. The

884 observed effects on the endothelial populations discarded the initial assumption that UC-

885 MSCs presented increased potential than DPSCs, since both CM presented comparable

886 stimulatory and protective effects on this population. Significant differences between the

887 two media performances were seldom observed and, whenever present, indicated

888 superior performance to DPSCs-CM supplemented groups. The variety of growth factors

889 and observed effects emphasises on the complementarity of functions between the

890 known and unknown factors that compose MSCs secretion cocktail [63]. This

891 complementarity is also well patent in some works, where the abolishment of one factor

892 only partially attenuate the effects displayed [58].

893 To the authors knowledge, this is the first comparative approach to the umbilical cord

894 and dental pulp derived stem/ stromal cells populations metabolomic and bioactive

895 secretion profiles. In summary, the present work provided insight on the metabolic profile

896 of UC-MSCs and DPSCs in culture during the serum-free conditioning process through

897 which MSCs conditioned media was obtained. Some differences were evidenced in the

898 metabolite dynamics on the CM from MSCs derived from the umbilical cord stroma and

899 from the dental pulp through the conditioning period (particularly on glucose

900 metabolism), but similar global metabolic profiles are suggested. Limited literature is

901 available to compare on the metabolic profiles of MSCs.



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902 More prominent differences are highlighted for the bioactive factors content of these

903 CMs, where FST, HGF, G-CSF, IL-8 and MCP-1 dominate in the UC-MSCs secretion,

904 while VEGF-A and FST are the most prominently produced by DPSCs.

905 Finally, the distinct secretory cocktail did not result in significantly different effects on

906 endothelial cell populations (proliferation, migration and tube formation capacity). The

907 apparent decreased chemotactic factors content of DPSCs was additionally refuted by

908 the comparable capacity to induce MSCs migration in vitro.

909

910 4. Acknowledgements

911 This research was supported by Programa Operacional Regional do Norte (ON.2 – O

912 Novo Norte), QREN, FEDER with the project “iBone Therapies: Terapias inovadoras

913 para a regeneração óssea”, ref. NORTE-01-0247-FEDER-003262, and by the program

914 COMPETE – Programa Operacional Factores de Competitividade, Projects PEst-

915 OE/AGR/UI0211/2011 and PEst-C/EME/UI0285/2013 funding from FCT. This research

916 was also supported by Programa Operacional Competitividade e Internacionalização

917 (P2020), Fundos Europeus Estruturais e de Investimento (FEEI) and FCT with the

918 project “BioMate – A novel bio-manufacturing system to produce bioactive scaffolds for

919 tissue engineering” with reference PTDC/EMS-SIS/7032/2014 and by COMPETE 2020,

920 from ANI – Projetos ID&T Empresas em Copromoção, Programas Operacionais POCI,

921 by the project “insitu.Biomas - Reinvent biomanufacturing systems by using an usability

922 approach for in situ clinic temporary implants fabrication” with the reference POCI-01-

923 0247-FEDER-017771.

924 This work received further financial support from the framework of QREN through Project

925 NORTE-07-0124-FEDER-000066. The Bruker Avance III 600 HD spectrometer was

926 purchased under the framework of QREN, through Project NORTE-07-0162-FEDER-

927 000048 and is part of the Portuguese NMR Network created with support of FCT through

928 Contract REDE/1517/RMN/2005, with funds from POCI 2010 (FEDER).



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929 Ana Rita Caseiro (SFRH/BD/101174/2014) acknowledges FCT, for financial support.

930 The authors acknowledge MF Gärtner and laboratory technicians A Rema, from the

931 Veterinary Pathology Laboratory – ICBAS, University of Porto, for their assistance in the

932 histopathologic analysis of subcutaneous tissue. The author also thanks Professor Elísio

933 Costa, from the Department of Biological Sciences of the Faculty of Pharmacy,

934 University of Porto, for his insight on the bioactive factors data statistical treatment.

935

936 5. Conflicts of Interest

937 The authors declare that there are no competing interests.

938

939 6. Supporting information captions

940 S1 Table: Statistically significant differences in Metabolites concentration (µM)

941 calculated from 1H-NMR spectra of unconditioned (Plain Medium), and UC-MSCs and

942 DPSCs conditioned media, after 24 and 48 hours. Significance of the results is indicated

943 according to P values with one, two, three or four of the symbols (*) corresponding to

944 0.01≤P<0.05; 0.001≤P< 0.01; 0.0001≤P<0.001 and P<0.0001, respectively; ns, not

945 significant.

946 S2 Table: Statistically significant differences on detected Bioactive factors (pg/mL)

947 calculated from Multiplexing LASER Bead Analysis of UC-MSCs and DPSCs

948 Conditioned Media after 24 and 48 hours of conditioning. Significance of the results is

949 indicated according to P values with one, two, three or four of the symbols (*)

950 corresponding to 0.01≤P<0.05; 0.001≤P<0.01; 0.0001≤P<0.001 and P<0.0001,

951 respectively; ns, not significant.

952 S3 Table: Significance of the results of the Presto Blue® Assay of UVECs, at 24, 48, 72

953 and 96 hours of expansion in control or CMs supplemented media, according to P values



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40

954 with one, two, three or four of the symbols (*) corresponding to 0.01≤P<0.05;

955 0.001≤P<0.01; 0.0001≤P<0.001 and P<0.0001, respectively; ns, not significant.

956 S4 Table: Significance of the results of the Apoptosis (Annexin-V/ PI) assay of UVECs,

957 after 48 hours of expansion in Complete, Control or CMs supplemented media,

958 according to P values with one, two, three or four of the symbols (*) corresponding to

959 0.01≤P<0.05; 0.001≤P<0.01; 0.0001≤P<0.001 and P<0.0001, respectively; ns, not

960 significant.

961

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1261

1262 * Corresponding author: Prof. Ana Colette Maurício - Departamento de Clínicas

1263 Veterinárias, Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do

1264 Porto (UP), Rua de Jorge Viterbo Ferreira, nº 228, 4050-313 Porto, Portugal; Mobile:

1265 +351 919 071 286, Phone: +351 220 428 000, Email: [email protected],

1266 [email protected]; ORCID-ID: https://orcid.org/0000-0002-0018-9363

1267

1268 e-mail contact:

1269 AR Caseiro: [email protected];

1270 SS Pedrosa: [email protected];

1271 G Ivanova: [email protected];

1272 MV Branquinho: [email protected];

1273 A Almeida: [email protected];

1274 F Faria: [email protected];

1275 I Amorim: [email protected];

1276 T Pereira: [email protected];

1277 AC Maurício: [email protected] , [email protected]

1278

1279 Authors Contributions

1280 Conceptualization: ARC, SSP, TP, ACM



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52

1281 Data curation: ARC, SSP, GI, MVB

1282 Formal analysis: ARC, SSP, GI, MVB, AA, FF, IA, TP, ACM

1283 Funding acquisition: ACM

1284 Investigation: ARC, SSP, GI, MVB, IA, TP, ACM

1285 Methodology: ARC, SSP, GI, MVB, AA, FF, IA, TP, ACM

1286 Project administration: ACM

1287 Software: ARC, SSP, MVB, IA

1288 Supervision: ACM

1289 Validation: ACM

1290 Writing – original draft: ARC, ACM

1291 Writing – review & editing: ACM

1292



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https://doi.org/10.1101/728550


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